2-Mercaptoethylamine antibodies

The use of a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody presence was found to result in a partially reduced antibody in which two disulfides were reduced to yield four thiols (Sun et al., 2005). This strategy can be used to retain a biospecific antibody construct for subsequent discrete conjugation at the hinge region between the heavy chains. 

Figure 1.77 Disulfide reducing agents such as 2-mercaptoethylamine can be used to cleave the disulfide bonds in the hinge region of antibody molecules. Either intact IgG molecules or F(ab )2 fragments may be reduced in this manner to yield monofunctional antigen binding fragments.

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction by-products by dialysis or gel filtration using a desalting resin. All buffers should contain 1-10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl-containing half antibody now may be used in conjugation protocols that use —SH-reactive heterobifunctional crosslinkers (Chapter 5, Section 1). 

To each milliliter of the antibody solution, add 6 mg of 2-mercaptoethylamine hydrochloride (final concentration is 50 mM). Mix to dissolve. 

Reduce the antibody starting material. Add 2-mercaptoethylamine HC1 (2-MEA, cysteamine HC1) to the antibody solution to a final concentration of 6-7 mg/mL (approx 53-62 mM) while stirring. Dithiothreitol (DTT) at a final concentration of 50 mM also can be used for reduction. 

Reduce the antibo by incubating in 50 mM 2-mercaptoethylamine for 1.5 h at 37 C. MEA obtained from Pierce (product 20408) comes in 6 mg lots, 50 mM equates to 6 mg/ml and the antibody can be added directly to the MEA. The total volume should be kept low to allow efficient separation on the desalting column. 

Mercaptoethylamine HQ (Pierce. 20408) Monoclonal antibody or pepsin-digested... 

The steps to reduce the antibodies followed the works of Mahnke et al [10]. The antibody was dissolved at a concentration of 10 mg/ml in 20 mM sodium phosphate buffer (PH7.5) containing 150 mM NaCl and 10 mM ethylene diamine tetraacetic acid (EDTA). Then 6 mg of 2-mercaptoethylamine (2-MEA) was added to the antibody solution. 

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